The fate of cells depends on their ability to sense and respond appropriately to their environment. The Jun Kinase (JNK) signal transduction pathway is activated in response to a wide variety of developmental and environmental signals to control fundamental cell behaviors, such as proliferation, differentiation, morphogenesis, and apoptosis, but the mechanisms that regulate the appropriate response to particular signals are poorly understood. Inappropriate activation of JNK signaling in humans can result in numerous disorders including chronic inflammation, cancer, and metabolic disease, while loss of JNK signaling early in development results in birth defects in mice. The overall objective of this proposal is to understand how JNK signaling is regulated to generate the appropriate responses in animal development and homeostasis. One way to regulate the appropriate response is to use unique combinations of signal transducers downstream from different signals. A candidate transducer that is selectively activated in certain JNK dependent processes in Drosophila is the Mixed Lineage Kinase (MLK), encoded by the slipper (slpr) locus. SLPR/MLK is an essential and specific regulator of tissue morphogenesis, which functions at the level of JNK Kinase Kinase (JNKKK) to stimulate JNK-dependent tissue reorganization. To investigate the molecular mechanisms by which SLPR/MLK regulates JNK signaling and tissue organization, we propose the following specific aims: (1) Characterization of essential functional domains of SLPR using phenotypic analysis of slpr mutant animals, coupled with expression of mutant, deleted, and chimeric JNKKK transgenes. (2) Identification of modifiers of JNK signaling by genetic screening for proteins that modify the defects associated with loss of SLPR function. (3) Determine the role of MSN Kinase and the PVR pathway in selective activation of SLPR during morphogenesis by in vitro binding assays and in vivo genetic analysis. The aims are designed to elucidate the requirement for MLK in a developmental system, the regulation of JNK signaling by MLK, and the selective recruitment of MLK protein complexes for specific JNK signaling outputs. These studies may ultimately suggest a molecular mechanism to manipulate JNK signaling therapeutically.